Antibodies are powerful tools for molecular and cellular analysis and for clinical diagnosis. The power comes from the considerable specificity of antibodies for their particular epitopes. This allows the detection or purification of a single species from among a very large background of competitors. The power of antibodies as a tool is enhanced by the ability to produce monoclonal antibodies against most desired antigens and to engineer antibodies further by recombinant DNA methods.
DNA molecules are also powerful tools for the analysis of molecules and cells and for clinical diagnosis. The power of DNA resides in the great specificity of stringent hybridization which allows about any unique DNA sequence to be detected specifically or, in principle, isolated. The power of DNA as a molecular tool is enhanced by the ability to make virtually any DNA sequence by automated methods and to amplify any DNA sequence from microscopic to macroscopic quantities by the polymerase chain reaction. Another very attractive feature of DNA is the great rigidity of short double helices. As judged by hydrodynamic criteria, DNA molecules of 30 to 60 base pairs are essentially rigid rods as far as flexure perpendicular to the helical axis is concerned. There is torsional flexibility, however, so that considerable rotation around the helical axis should be possible.
Many plausible mechanisms for environmental modulation of cell action or for cell--cell intimacy are based on alterations of distances between proteins at the cell surface. It is difficult to study or manipulate these distances because some of the molecules of interest are very rare, and most of the distances are too long to be observed by ordinary physical or chemical techniques.
One approach to solving these problems has been to use bispecific antibodies. In addition to hybrid hybridoma or "quadroma" generation (Milstein and Cuello, 1983), bispecific antibodies may also be created via chemical cross-linkage. Of the many methods available, disulfide exchange between reduced hinge region sulfides of antibody Fab' fragments may prove to be the most efficient in terms of both yield and defined homogeneity of product (Brennan et al., 1985). However, this and most other current cross-linking methods lack the option of varying the relative 3-dimensional spatial orientation of the 2 antigen combining sites. Of note, though, is the current strategy employed by Kostelny et al. (1992) in which they expressed 2 different Fab'-leucine zipper (derivatives of Fos and Jun) fusion proteins in mouse myeloma cells to form 2 separate F(ab'-zipper).sub.2 homodimers, reduced them in vitro, and then mixed them to efficiently form F(ab'zipper).sub.2 heterodimers, to provide a resultant specific antibody of some defined structure. The proper functioning of a cell depends on the interaction of the various components on the surface of that cell. It has been shown that CD4 may act to increase affinity of the T cell for class II MHC expressing cells by direct binding to class II (Doyle and Strominger, 1987). Moreover, it may also act synergistically with the TCR in a signaling capacity (Fleischer and Schrezenmeier, 1988). Various other studies have directly shown that subsets of CD4 and the TCR are in close association with each other on the cell surface (For example, see Gallagher et al., 1989; Chuck et al., 1990). Still others have demonstrated that CD4 and the TCR may bind to the same class II MHC molecule (Miceli et al., 1991). These data imply that CD4 is probably both physically and functionally linked to the TCR.
Many studies have shown that T cells may be triggered via certain anti-receptor monoclonal antibodies, including those against CD4 and the TCR (for example, see Haque et al., 1987; Janeway et al., 1987).
In an approach to examine the association between CD4 and the T cell receptor (TCR) on the cell surface. T cells were triggered with hetero-cross-linked anti-CD4 and anti-TCR antibodies. It was found that the heterocombination triggers much more efficiently than the homo-combinations unlinked antibodies (Ledbetter et al., 1988). These results were used as indirect evidence for functional as well as physical associations between 2 T cell surface molecules. However, no one has yet investigated the actual role that distance may play in effecting such hetero-cross-links, since long range cross-linkers do not exist.
In the previous experiments in which T cells were triggered via the CD4:TCR complex, workers used either solid state supports such as microbeads, secondary antibodies or chemical cross-linkers to cross-link anti-CD4 to anti-TCR. In such studies the relative positions of the two different triggering antibodies were largely unknown. Further, in the study using microbeads it was impossible to tell how many receptors were aggregated by the beads at the cell contact point. Therefore, the functionality of the CD4-TCR physical association has yet to be explored. Relative position information would be useful in dissecting the geometry of the putative CD4:TCR activation complex since the distance separating CD4 and TCR cannot be accurately determined even by energy transfer measurements. One question is whether CD4 needs to be close to the TCR, i.e., act as a co-receptor during the process of antigen recognition (Janeway, 1988) which may include presentation of p56.sup.1ck and other kinases to CD3:TCR (Haughn et al., 1992), or whether it can just as well assert its synergistic action over a longer distance.
Accordingly, there exists a need for a better technique for precisely measuring the distance between proteins on cells and for a different means for varying and controlling the three dimensional spatial separation of combining sites in bispecific antibodies.
Further, most immunoassays do not allow for the detection of a single molecule in a sample. Therefore, a need exists for more methods that can accurately detect single molecules in a sample.
Also there exists several instances where the polymerase chain reaction has been combined with other immuno techniques to identify various entities such as viruses and bacteria in a sample. However, most of these techniques as well as other common immunoassays are capable of detecting the presence of only a single analyte in a sample. Therefore, there also exists a need for an assay which will coincidentally detect more than one analyte such as pairs of viral antigens in a single sample.